Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vβ21.3+ activation in multi-inflammatory syndrome in children

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vβ21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vβ21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vβ21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

There are 2 samples without intravenous immunoglobin (IVIG) or glucocorticoid pre-treatment, 1 pre-treated with IVIG and 2 with glucocorticoid.(E) Expression levels of NCAM1 (CD56), FCGR3A (CD16), KLRD1, CCR7, CX3CR1 in all clusters were shown in the violin plots.(F) CD16-NK cells were re-clustered and cluster 0, 1, 2, 3, 6 were selected as true CD16-NK cells for further comparison between admission and follow-up group.IL18 showed a significant change again.(G) Markers of SOX4, KIT, NCAM1, KLRC1 in the CD16-NK cells.Note high SOX4 and KIT expressing cells in cluster 4 and 5 of (F), markers of hematopoietic stem cell.

SupplementaryFigure 1
Correlation between cytokines and clinical test markers, temporal changes in cytokines in MIS-C.Related to Figure 2. (A) Correlation between IL1-α and troponin.(B) Correlation between IL-1β and troponin.(C) Correlation between HGF and neutrophil count.(D) Correlation between IL-1α and D-dimer.(E) Correlation between IL1β and D-dimer.(F) Correlation between MCP-1 and D-dimer.Pearson correlation test was used: * = p<0.05,** = p<0.01.(G) Plasma levels of TNF-α, M-CSF, IL2RA, and IFN-γ for six MIS-C patients at acute, recovery (2-4 days after PICU admission) and follow-up (approximately one month after discharge) time points.Source data are provided as a Source Data file.

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and unmixing of CyToF data.Related to Figure 3. (A) Plotting of events over time.(B) Gating to remove beads.(C)-(F) Gating of single live cells using event length, width, and residual parameters followed by iridium DNA dye and rhodium live/dead dye.(G)-(I) Example of unmixing of pooled samples, labelled with CD45-89Y, CD45-195Pt, CD45-98Pt and B2M/ CD298-106Cd.gating of different T cell types using Flowjo software.Related to Figure 3. (A) Gating using CD3 and CD19 parameters to gate CD3+ T cells.(B) Excluding NK cells by gating on CD56-negative events.(C) Excluding γδ T-cells by gating on TCRγδ-negative events.(D) Excluding MAIT cells by gating on TCR-Vα7.2 negative events.(E) Gating of CD4+ and CD8+ T cells from clean CD3+ T cells selected as shown in panels A-D.(F) Gating of CD25high CD127low Treg cells from CD4+ T cells.(G) Gating of CCR4+ CCR6+ Th17 cells from non-Treg cells.(H) Gating of CXCR3+ Th1 cells and CCR4+ Th2 cells from negative population in (F).(I and J) Gating of TCR-Vβ 21.3+ events from Th1 and Treg, respectively.Analysis of T cell panel CyToF data by CATALYST package.Related to Figure 3. (A) MDS plotting of samples by cell type markers.(B) Heatmap of cell population abundance comparing MIS-C acute with healthy volunteer samples.(C and D) Expression levels of CD95 (C) or IL-18R (D) in different populations of immune cells in PBMCs from acute MIS-C patients or all other samples; cell populations on the Y axis were ordered by Euclidean distance of CD95 and CD-18R expression in (C) and (D), respectively.Additional data from CyToF analysis using the T-cell panel of antibodies.Related to Figure 3. (A) opt-tSNE projections showing levels of CD4, CD8, TIM3, LAG3, CD25, CD57 markers in the non-naïve T cell populations from the indicated groups of subjects.(B) op-tSNE plotting of non-naïve T cell density in the infectious disease group split by sepsis and non-sepsis conditions.(C) Frequency of TCR Vβ 21.3+ cells in CD4 T cells.(D) Frequency of TCR Vβ 21.3+ cells in CD8 T cells.(E) Frequency of TCR Vβ 21.3+ cells in DN T cells.(F) Percentage of TCR Vβ21.3+T cells in different subsets of CD4 T cells (naïve, central memory, effector memory, terminal effector).(G)-(J) Frequency of Th1, Th2, Th17 and Treg in CD4 T cells.(K) Frequency of CD38+ HLA-DR+ activated T cells in DN T cells.(L) Frequency of CD38+ HLA-DR+ activated T cells in MAIT cells.(M) Frequency of CD38+ HLA-DR+ activated T cells in γδT cells.Ordinary one-way ANOVA was conducted, with Dunnett's multiple comparisons test comparing other groups with MIS-C group: * = p<0.05,** = p<0.01,*** = p<0.001,**** = p<0.0001.Source data are provided as a Source Data file.
analysis using additional markers and ASC staining.Related to Figure 4 and 5. (A) ROC analysis of IL-10 comparing MIS-C samples with respiratory inflammation group as control.(B) ROC analysis of IL-10 comparing MIS-C samples with respiratory inflammation group as control.(C)-(E) ROC analysis of IL-18R, CD38 and ICOS in non-naïve CD8 T cells comparing MIS-C samples with infection samples as control.(F) Representative images from samples where ASC was stained in whole blood fixed with cytodelics kits, including positive control (healthy subject blood samples stimulated with LPS plus ATP to induce NLRP3 activation), MIS-C sample (n=3), MIS-C discharge sample (n=2) and acute COVID-19 sample(n=1).ASC specks can be seen in the positive samples (pointed by white arrow), as well as acute COVID-19 samples, but not MIS-C or MIS-C follow-up samples.Blue staining: DAPI, green staining: ASC.Source data are provided as a Source Data file.
scRNA-seq analysis.Related to Figure 6.(A) Plot showing the abundance of all TCR Vβ subtypes in non-naïve CD4 and cytotoxic CD8 T cells from five MIS-C patients, showing samples collected during the acute phase of disease and at their follow-up visit approximately one month after hospital discharge.Patient code is shown on the X axis, with suffix T1 or T3 indicating acute or follow up stage respectively.(B) Vβ and j chain pairing in acute MIS-C cytotoxic CD8 T cells.(C) Top 20 genes differentially expressed between TCR Vβ21.3+ and TCR Vβ21.3-populations in central memory CD4 T (left) and cytotoxic CD8 T (right) cells.(D) Expression of M1 and M2 markers in acute MIS-C monocytes.